The major goal of this proposal is to clarify the molecular events that control the differentiation of motor neurons in the developing spinal cord. Inductive signals mediated by the secreted factor Sonic hedgehog (Shh) have been shown to initiate the differentiation of neural progenitor cells into motor neurons by activating or repressing the expression of transcription factors, most notably homeodomain proteins. It is likely that other extrinsic signals also contribute to the specification of motor neuron fate, and many of the transcriptional regulatory events that occur downstream of Shh signaling remain poorly defined. This proposal will therefore focus on three aspects of spinal motor neuron differentiation. First, the molecular pathway that directs progenitor cells to a generic motor neuron identity will be defined. In particular, the respective contributions of two major classes of transcription factors, homeodomain and basic helix loop helix proteins, will be studied using in vitro and in vivo assays of gene function in chick and mouse spinal cord. Second, the molecular steps that direct the formation of distinct columnar subclasses of motor neurons will be examined in transgenic chicks and mice, focusing initially on the roles of two classes of extrinsic inductive signals, Shh and BMPs, on the generation of motor neuron diversity at thoracic levels of the spinal cord. Third, the molecular mechanisms that control the projection of motor axons to their targets in the periphery will be examined in transgenic chicks and mice. In particular, experiments will attempt to relate the transcriptional control of motor neuron identity to the expression of cell surface and cytoplasmic effector proteins that direct motor axons as they enter the developing limb. Together, these studies are intended to outline the assembly of a molecular pathway that links early inductive signals, transcription factors and axon guidance molecules to the formation of motor connections in the periphery. The selective degeneration of motor neurons underlies many neurological disorders, notably the spinal muscular atrophies and amyotrophic lateral sclerosis. The selectivity of motor neuron degeneration that is a characteristic of these diseases remains unexplained. Defining the molecular steps that control motor neuron development may therefore provide insight into the basis of these disorders, and in the long term lead to the design of novel strategies for their treatment. In addition, the study of the molecular basis of motor axon outgrowth may aid the development of more effective therapies for recovery of motor function after traumatic injury to the spinal cord.